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Altenbuchner et al., xe2x80x9cPositive selection vectors based on palindromic DNA sequencesxe2x80x9d Methods Enzymol. 216, 457-466 (1992).
Bernard et al., xe2x80x9cNew ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markersxe2x80x9d Gene 148, 71-74 (1994).
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Henrich, B. and Schmidtberger, B., xe2x80x9cPositive-selection vector with enhanced lytic potential based on a variant of phi X174 phage gene Exe2x80x9d Gene 154, 51-54 (1995).
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Messing et al., xe2x80x9cFilamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitroxe2x80x9d Proc. Natl. Acad. Sci. 79, 3642-3646 (1977).
Mullis, K. B. and Faloona, F. A., xe2x80x9cSpecific synthesis of DNA in vitro via polymerase-catalyzed chain reactionxe2x80x9d 1987, Methods Enzymol. 155, 335-350 (1987).
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Saiki et al., xe2x80x9cEnzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemiaxe2x80x9d Science 230, 1350-1354 (1985).
Yanisch-Perron et al., xe2x80x9cImproved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 pUC19 vectorsxe2x80x9d Gene 33, 103-119 (1985).
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The present invention relates to a positive selection vector system for direct cloning of PCR amplified nucleic acids. The invention involves insertional reconstruction of a reporter or of a regulatory gene. The invention describes reduction of exonuclease-induced false positive clones in a cloning experiment.
Polymerase chain reaction or PCR (Saiki et al., 1985, Science 230, 1350-1354; Mullis and Faloona, 1987, Method Enzymol. 155, 335-350; U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,965,188) is a milestone technological development in the field of molecular biology and genetic engineering. For amplification of a target nucleic acid PCR uses a polymerase, target sequence-specific forward and reverse primers, deoxynucleotides and a minute amount of target nucleic acid as the template. Repeated cycles of denaturation of double-stranded DNA followed by primer annealing and primer extension achieve an exponential amplification of the target DNA sequence.
The PCR product itself could be used for diagnosis, quantitation of the template, direct sequencing and some other applications (U.S. Pat. Nos. 5,856,144; 5,487,993 and 5,891,687). However, for applications such as mutation analysis, sequencing, gene expression, identification of polymorphic transcripts, making RNA probes etc., usually a large quantity of DNA is needed. Thus it is necessary to isolate a bacterial clone carrying the PCR generated target DNA fragment in a vector. Different methods for cloning PCR generated DNA fragments have been described. One such method involves incorporation of restriction endonuclease cleavage sites near the 5xe2x80x2 end of the PCR primers and the PCR product thus obtained is subjected to purification, restriction digestion with the respective endonuclease followed by ligation into a compatible vector, transformation and identification of the bacterial clone carrying the PCR fragment (Kaufmann and Evans, 1990, BioTechniques 9, 304-306).
The most common method for cloning a PCR product utilizes the nontemplate-dependent terminal transferase or extendase activity of Taq DNA polymerase, which usually produces a dAMP (deoxyadenosine monophosphate) overhang at the 3xe2x80x2 end of the PCR amplified DNA fragment (Clark, 1988, Nucl. Acid Res. 16, 9677-9686; Hu, 1993, DNA Cell Biol. 12, 763-770). The PCR product thus obtained is ligated into a linearized vector carrying a dTMP (deoxythymidine monophosphate) overhang at the 3xe2x80x2 end (U.S. Pat. No. 5,487,993; Mead et al., 1991, BioTechniques 9, 657-663; Holton and Graham, 1991, Nucl. Acids Res. 19, 1156). A similar strategy has been used when Taq polymerase generated PCR fragments carrying dAMP overhang at the 3xe2x80x2 end are ligated into a linearized vector carrying an inosine or uracil overhang at the 3xe2x80x2 end (U.S. Pat. No. 5,856,144).
The above-mentioned vectors lack the positive selection capability. Thus upon transformation, all host cells carrying either the recombinant vector (containing an insert) or the nonrecombinant vector (containing no insert DNA) grow in the desired medium at an equal growth rate. To differentiate between a host cell carrying only the nonrecombinant vector from the host cell carrying the recombinant vector, DNA fragment is usually inserted into a chromogenic gene, the product of which is inactivated thus rendering the recombinant colony white in a chromogenic medium. When the chromogenic gene is lacZ, the transformant carrying the nonrecombinant vector turns blue in the presence of X-gal, the substrate for the lacZ gene product xcex2-galactosidase (Messing et al., 1977, Proc. Natl. Acad. Sci. 79, 3642-3646; Norrander et al., 1983, Gene 26, 101-106; Yanisch-Perron et al., 1985, Gene 33, 103-119). When the number of recombinant colonies are low and nonrecombinant colonies are high in a plate, then it becomes very difficult to differentiate the recombinant colonies from the non-recombinant colonies. High number of colonies also lead to contamination between the recombinant and nonrecombinant colonies. Insertion of a small DNA fragment sometimes can generate pale blue recombinant clones, which may not be differentiated from the pale blue nonrecombinant clones arising from nonuniform distribution of Xgal, especially when Xgal is spread on the surface of medium.
To ameliorate the problems associated with the chromogenic selection of the recombinant clones many vectors have been developed with positive selection capability allowing only the recombinant clones to grow in a selection medium. Most of these positive selection vectors have been developed based on insertional inactivation of lethal genes (Pierce et al., 1992, Proc. Natl. Acad. Sci. 89, 2056-2060; Henrich and Plapp, 1986, Gene 42, 345-349; Henrich and Schmidtberger, 1995, Gene 154, 51-54; Bernard et al., 1994, Gene 148, 71-74; Kuhn et al., 1986, Gene 42, 253-263; U.S. Pat. No. 5,910,438; U.S. Pat. No. 5,891,687). A vector system based on abolition of sensitivity towards metabolite has also been described (Kast, 1994, Gene 138,109-114). Vectors have also been constructed based on selection by means of DNA-degrading or RNA-degrading enzymes (Yazynin et al., 1996, Gene 169, 131-132; Ahrenhotz et al., 1994, Appl. Environ. Microbiol. 60, 3746-3751) as well as based on selection by destruction of long palindromic DNA sequences (Altenbuchner et al., 1992, Methods Enzymol. 216, 457-466).
The presently available positive selection vectors as well as other cloning vectors are associated with many disadvantages. An inherent problem of a vector with a lethal or a chromogenic gene is a high number of false positive clones, i.e., clones without any insert. The false positive clones could be revertants arising out of dominant mutations in the lethal or chromogenic gene rendering it inactive. However, the biggest disadvantage of every cloning system available today is the exonuclease-induced generation of false positive clones. The reagents used in restriction digestion, PCR and ligation, such as restriction enzymes, polymerases and ligases, are usually contaminated with exonucleases, which are seldom completely removed from larger lots of commercial preparations. Exonuclease digestion deletes some nucleotide bases from the cloning site in the chromogenic or lethal gene in a linearized vector DNA. Thus recircularization of such vectors results in inactivation of the chromogenic or lethal genes, and upon transformation, these recircularized vectors give false positive transformant clones. A palindromic sequence could also be destroyed by exonuclease digestion resulting in generation of false positive clones.
Insertion of a small DNA fragment in frame with the nucleotide sequence of the lethal gene or the chromogenic gene may in some cases not alter the function of the lethal or chromogenic gene, thus making it impossible to clone such small DNA fragments. Furthermore, when cloning of a small DNA fragment results in diminished function of the lethal gene, which nevertheless remains functional, then the recombinant clones grow at a reduced rate in case of positive selection vectors, and these clones could be confused with the non-recombinant clones growing because of diminished selection pressure due to, for example, long period of incubation.
A further disadvantage of the vectors based on lethal genes is that it may require a complex medium to activate the selection mechanism (Kast, 1994, Gene 138, 109-114). The positive selection vectors carrying lethal or chromogenic genes also require special host cells for transformation, e.g., CcdB based vectors require Fxe2x88x92 host cells (U.S. Pat. No. 5,910,438), CAP based vectors require adenyl-cyclase positive host cells (U.S. Pat. No. 5,891,687) and lacZ based vectors require lacxe2x88x92 host cells (Messing et al., 1977, Proc. Natl. Acad. Sci. 79, 3642-3646; Norrander et al., 1983, Gene 26, 101-106; Yanisch-Perron et al., 1985, Gene 33, 103-119). A special regulatory system, usually lacI or CI repressor system (U.S. Pat. No. 5,910,438; Pierce et al., 1992, Proc. Natl. Acad. Sci. 89, 2056-2060), has also to be in place to prevent the expression of the lethal gene in the host cell during the preparation of vector DNA.
The primary object of the present invention is to develop a simple cloning and/or sequencing vector having the capability of positive selection thus allowing only the recombinant clones (carrying an insert DNA) to grow in a selection medium, whereas, the non-recombinant clones (carrying no insert DNA) would not grow. The vector could also be used as a positive selection expression vector.
The particular object of the present invention is to eliminate or greatly reduce the generation of false positive clones associated with all the presently available cloning systems. Especial emphasis is given to the elimination of exonuclease-induced false positive clones. Thus the present invention aims to apply the principle of insertional reconstruction of a reporter gene or a regulatory gene controlling the expression of a reporter gene. It was aimed to develop a positive selection vector based on insertional reconstruction of an antibiotic resistance reporter gene, which carries a dominant negative mutation at its 5xe2x80x2 or 3xe2x80x2 end. Thus upon transformation non-recombinant clones will not grow in presence of the respective antibiotic. Insertional reconstruction allows correction of the mutation in the antibiotic resistance reporter gene. Thus upon transformation of a host cell the reconstructed reporter gene produces functionally active antibiotic resistance reporter gene protein thus allowing the host cell to grow in a specific selection medium containing the respective antibiotic.
Use of the principle of reconstruction of a reporter gene should also greatly reduce, if not eliminate, revertants because firstly, probability of spontaneous mutational reconstruction of the wild-type reporter or regulatory gene is minimal, and secondly, any mutation in the coding sequence of the reporter or regulatory gene would rather negatively affect the function of the respective gene protein.
A vector system based on antibiotic resistance gene as the reporter gene should also eliminate the need of any special type of host cells.
The elimination of the disadvantages associated with the presently available vectors is greatly desirable. A vector system based on reporter gene reconstruction will mostly eliminate these disadvantages and hence will be a substantial technological achievement.
The present invention relates to a strategy for developing positive selection vectors based on reconstruction of a reporter gene or of a regulatory gene controlling the expression of a reporter gene. The invention also describes the use of such vectors for direct cloning of PCR products. As an example of application of the strategy, a positive selection vector pRGR1Ap has been developed. When the last (position 286) amino acid tryptophan (encoded by 5xe2x80x2-TGG-3xe2x80x2) of ampicillin resistance gene xcex2-lactamase is replaced by valine (encoded by 5xe2x80x2-GTG-3xe2x80x2) xcex2-lactamase becomes functionally inactive. The sequence 5xe2x80x2-GTG-3xe2x80x2 is a part of the PmlI restriction endonuclease cleavage site 5xe2x80x2-CACGTG-3xe2x80x2, which is a unique cloning site in this vector. Thus upon PmlI restriction endonuclease cleavage 5xe2x80x2-CAC-3xe2x80x2 and 5xe2x80x2-GTG-3xe2x80x2 are created at the 3xe2x80x2 and 5xe2x80x2 ends respectively of the linearized vector. A PCR primer carrying the nucleotides 5xe2x80x2-TGGTAA-3xe2x80x2 at its 5xe2x80x2 end is used in PCR. When the resulting blunt-ended PCR products thus obtained are ligated to the vector the reporter ampicillin resistance gene is reconstructed correcting the mutation. The nucleotides 5xe2x80x2-TAA-3xe2x80x2 constitute the stop codon for the xcex2-lactamase gene. Subsequent transformation of a host cell with the recombinant vector (carrying an insert DNA) produces functionally active xcex2-lactamase, which confers resistance to ampicillin.
Restriction sites of ClaI, EcoRV, NarI, NdeI, NotI and SfiI have been introduced in this vector for easy extraction of the insert. The NarI site in the xcex2-lactamase gene does not change the amino acid sequence of xcex2-lactamase. Introduction of EcoRV restriction site in xcex2-lactamase gene (bla) changes glutamic acid at position 277 into aspartic acid, whereas, the restriction site NdeI changes isoleucine into methionine at position 275, and glutamine into histidine at position 274. The restriction site ClaI in xcex2-lactamase gene (bla) replaces methionine at position 268 with isoleucine. The mutations introduced by restriction sites NarI, EcoRV, NdeI and ClaI do not have any significant effect on the function of xcex2-lactamase. Another vector pRGR2Ap, which is similar to pRGR1Ap, has been described. The vector pRGR2Ap contains pUC origin of replication, M13 origin of replication and T7 phage promoter.